The metal ion binding properties of calreticulin modulate its conformational flexibility and thermal stability

Calreticulin (CRT) is a soluble chaperone involved in the conformational maturation of glycoproteins in the endoplasmic reticulum. Using biochemical and biophysical techniques including circular dichroism, proteolysis, and analytical ultracentrifugation, we have determined the effects of calcium and zinc ions on the structural properties of human CRT. Circular dichroism analysis has shown that the binding of calcium and zinc ions to CRT induces no significant changes in the secondary structure of the protein but affects in very distinct ways the local tertiary packing of these elements. More specifically, these studies have revealed that CRT adopts a more rigid and thermally stable structure upon binding calcium ions and a more loosely packed and thermally destabilized structure upon binding zinc ions. Consistent with these results, proteolysis experiments demonstrated that the intrinsic conformational flexibility of CRT can be modulated toward either a decrease or an increase in susceptibility to cleavage by chymotrypsin upon binding calcium or zinc ions, respectively. Results from sedimentation analysis indicated that the global three-dimensional structure of CRT is essentially unchanged upon binding calcium ions. In marked contrast, CRT self-associates reversibly to form dimers upon binding zinc ions. Collectively, our results provide evidence that calcium and zinc ions induce strikingly different changes in the biochemical and structural properties of CRT.     

NMR structures of 36 and 73-residue fragments of the calreticulin P-domain

Calreticulin (CRT) is an abundant, soluble molecular chaperone of the endoplasmic reticulum. Similar to its membrane-bound homolog calnexin (CNX), it is a lectin that promotes the folding of proteins carrying N-linked glycans. Both proteins cooperate with an associated co-chaperone, the thiol-disulfide oxidoreductase ERp57. This enzyme catalyzes the formation of disulfide bonds in CNX and CRT-bound glycoprotein substrates. Previously, we solved the NMR structure of the central proline-rich P-domain of CRT comprising residues 189-288. This structure shows an extended hairpin topology, with three short anti-parallel beta-sheets, three small hydrophobic clusters, and one helical turn at the tip of the hairpin. We further demonstrated that the residues 225-251 at the tip of the CRT P-domain are involved in direct contacts with ERp57. Here, we show that the CRT P-domain fragment CRT(221-256) constitutes an autonomous folding unit, and has a structure highly similar to that of the corresponding region in CRT(189-288). Of the 36 residues present in CRT(221-256), 32 form a well-structured core, making this fragment one of the smallest known natural sequences to form a stable non-helical fold in the absence of disulfide bonds or tightly bound metal ions. CRT(221-256) comprises all the residues of the intact P-domain that were shown to interact with ERp57. Isothermal titration microcalorimetry (ITC) now showed affinity of this fragment for ERp57 similar to that of the intact P-domain, demonstrating that CRT(221-256) may be used as a low molecular mass mimic of CRT for further investigations of the interaction with ERp57. We also solved the NMR structure of the 73-residue fragment CRT(189-261), in which the tip of the hairpin and the first beta-sheet are well structured, but the residues 189-213 are disordered, presumably due to lack of stabilizing interactions across the hairpin.     

Interdomain cooperativity of calmodulin bound to melittin preferentially increases calcium affinity of sites I and II

Calmodulin (CaM) is the primary transducer of calcium fluxes in eukaryotic cells. Its two domains allosterically regulate myriad target proteins through calcium-linked association and conformational change. Many of these proteins have a basic amphipathic alpha-helix (BAA) motif that binds one or both CaM domains. Previously, we demonstrated domain-specific binding of melittin, a model BAA peptide, to Paramecium CaM (PCaM): C-domain mutations altered the interaction with melittin, whereas N-domain mutations had no discernable effect. Here, we report on the use of fluorescence and NMR spectroscopy to measure the domain-specific association of melittin with calcium-saturated ((Ca(2+))(4)-PCaM) or calcium-depleted (apo) PCaM, which has enabled us to determine the free energies of calcium binding to the PCaM-melittin complex, and to estimate interdomain cooperativity. Under apo conditions, melittin associated with each PCaM domain fragment (PCaM(1-80) and PCaM(76-148)), as well as with the C-domain of full-length PCaM (PCaM(1-148)). In the presence of calcium, all of these interactions were again observed, in addition to which an association with the N-domain of (Ca(2+))(4)-PCaM(1-148) occurred. This new association was made possible by the fact that melittin changed the calcium-binding preferences for the domains from sequential (C > N) to concomitant, decreasing the median ligand activity of calcium toward the N-domain 10-fold more than that observed for the C-domain. This selectivity may be explained by a free energy of cooperativity of -3 kcal/mol between the N- and C-domains. This study demonstrates multiple domain-selective differences in the interactions between melittin and PCaM. Our findings support a model that may apply more generally to ion channels that associate with the C-domain of CaM under low (resting) calcium conditions, but rearrange when calcium binding triggers an association of the N- domain with the channel.     

Probing the three-dimensional structure of human calreticulin

Calreticulin (CRT) is an abundant soluble protein of the endoplasmic reticulum lumen that functions as a molecular chaperone for nascent glycoproteins. We have probed the three-dimensional structure of human CRT using a series of biochemical and biophysical approaches in an effort to understand the molecular basis of its chaperone function. Sedimentation analysis and chemical cross-linking experiments showed that CRT is monodisperse and monomeric in solution with a molecular mass (MW) of 46 +/- 1 kDa. This MW value together with a sedimentation coefficient, s(o)(20,w), of 2.71 S yielded a frictional ratio, f/f(0), of 1.65. Assuming CRT to be a prolate ellipsoid, we calculated an apparent length of 29.8 nm and diameter of 2.44 nm consistent with an asymmetric elongated molecule. These hydrodynamic dimensions account for the apparent anomalous elution position of CRT on gel filtration columns. Far-UV circular dichroism experiments showed that CRT has a cooperative thermal denaturation transition with a midpoint temperature of 42.5 degrees C suggesting a marginally stable structure. Proteolysis experiments showed that the highly acidic segment at the C-terminus of CRT is most susceptible to digest, consistent with the absence of a well-defined polypeptide backbone structure in this region of the protein. Temperature-dependent proteolysis with thermolysin revealed a stable core region within the N- and P-domains. A stable fragment encompassing most of the P-domain was also identified in the thermolytic mixture. Collectively, our results suggest that CRT is likely to be a flexible molecule in solution which may be important for its chaperone function.     

Target recognition by calmodulin: dissecting the kinetics and affinity of interaction using short peptide sequences.

The interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N-terminal target residues and the C-domain of calmodulin, and between the C-terminal target residues and the N-domain of calmodulin (Ikura M et al., 1992, Science 256:632-638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C-domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WF10 (residues 1-10 of M13) binds to CaM with Kd approximately 1 microM; peptide FW10 (residues 9-18 of M13, with Phe-17-->Trp substitution) binds to CaM with Kd approximately 100 microM. The effect of peptide WF10 on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C-domain, forming a stable half-saturated complex, Ca2-CaM-peptide, and confirming the functional importance of the interaction of this sequence with the C-domain. Stopped-flow studies show that the EGTA-induced dissociation of WF10 from Ca4-CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half-saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N-terminal target residues with the C-domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N- and C-domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin-dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C-domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.     

Zinc and calcium ions cooperatively modulate ADAMTS13 activity

ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) multimers. The metal ion dependence of ADAMTS13 activity was examined with multimeric VWF and a fluorescent peptide substrate based on Asp(1596)-Arg(1668) of the VWF A2 domain, FRETS-VWF73. ADAMTS13 activity in citrate-anticoagulated plasma was enhanced approximately 2-fold by zinc ions, approximately 3-fold by calcium ions, and approximately 6-fold by both ions, suggesting cooperative activation. Cleavage of VWF by recombinant ADAMTS13 was activated up to approximately 200-fold by zinc ions (K(D) (app) approximately 0.5 microM), calcium ions (K(D) (app) approximately 4.8 microM), and barium ions (K(D) (app) approximately 1.7 mM). Barium ions stimulated ADAMTS13 activity in citrated plasma but not in citrate-free plasma. Therefore, the stimulation by barium ions of ADAMTS13 in citrated plasma appears to reflect the release of chelated calcium and zinc ions from complexes with citrate. At optimal zinc and calcium concentrations, ADAMTS13 cleaved VWF with a K(m) (app) of 3.7 +/- 1.4 microg/ml (approximately 15 nM for VWF subunits), which is comparable with the plasma VWF concentration of 5-10 microg/ml. ADAMTS13 could cleave approximately 14% of VWF pretreated with guanidine HCl, suggesting that this substrate is heterogeneous in susceptibility to proteolysis. ADAMTS13 cleaved FRETS-VWF73 with a K(m) (app) of 3.2 +/- 1.1 microM, consistent with an approximately 200-fold decrease in affinity compared with VWF. ADAMTS13 cleaved VWF and FRETS-VWF73 with roughly comparable catalytic efficiency of 55 microM(-1) min(-1) and 18 microM(-1) min(-1), respectively. The striking preference of ADAMTS13 for VWF suggests that substrate recognition depends on structural features or exosites on multimeric VWF that are missing from FRETS-VWF73.     

The solution structure of apocalmodulin from Saccharomyces cerevisiae implies a mechanism for its unique Ca2+ binding property

We have determined the solution structure of calmodulin (CaM) from yeast (Saccharomyces cerevisiae) (yCaM) in the apo state by using NMR spectroscopy. yCaM is 60% identical in its amino acid sequence with other CaMs, and exhibits its unique biological features. yCaM consists of two similar globular domains (N- and C-domain) containing three Ca(2+)-binding motifs, EF-hands, in accordance with the observed 3 mol of Ca(2+) binding. In the solution structure of yCaM, the conformation of the N-domain conforms well to the one of the expressed N-terminal half-domains of yCaM [Ishida, H., et al. (2000) Biochemistry 39, 13660-13668]. The conformation of the C-domain basically consists of a pair of helix-loop-helix motifs, though a segment corresponding to the forth Ca(2+)-binding site of CaM deviates in its primary structure from a typical EF-hand motif and loses the ability to bind Ca(2+). Thus, the resulting conformation of each domain is essentially identical to the corresponding domain of CaM in the apo state. A flexible linker connects the two domains as observed for CaM. Any evidence for the previously reported interdomain interaction in yCaM was not observed in the solution structure of the apo state. Hence, the interdomain interaction possibly occurs in the course of Ca(2+) binding and generates a cooperative Ca(2+) binding among all three sites. Preliminary studies on a mutant protein of yCaM, E104Q, revealed that the Ca(2+)-bound N-domain interacts with the apo C-domain and induces a large conformational change in the C-domain.     

Expression of calreticulin in Escherichia coli and identification of its Ca2+ binding domains

Recombinant calreticulin and discrete domains of calreticulin were expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and their Ca2+ binding properties were determined. Native calreticulin bound 1 mol of Ca2+/mol of protein with high affinity, and also bound approximately 20 mol of Ca2+/mol of protein with low affinity. Both Ca2+ binding sites were present in the recombinant calreticulin indicating that proper folding of the protein was achieved using this system. Calreticulin is structurally divided into three distinct domains: the N-domain encompassing the first 200 residues; the P-domain which is enriched in proline residues (residue 187-317); and the C-domain which covers the carboxyl-terminal quarter of the protein (residues 310-401), and contains a high concentration of acidic residues. These domains were expressed in E. coli, isolated, and purified, and their Ca2+ binding properties were analyzed. The C-domain bound approximately 18 mol of Ca2+/mol of protein with a dissociation constant of approximately 2 mM. The P-domain bound approximately 0.6-1 mol of Ca2+/mol of protein with a dissociation constant of approximately 10 microM. The P-domain and the C-domain, when expressed together as the P+C-domain, bound Ca2+ with both high affinity and low affinity, reminiscent of both full length recombinant calreticulin and native calreticulin. In contrast the N-domain, did not bind any detectable amount of 45Ca2+. We conclude that calreticulin has two quite distinct types of Ca2+ binding sites, and that these sites are in different structural regions of the molecule. The P-domain binds Ca2+ with high affinity and low capacity, whereas the C-domain binds Ca2+ with low affinity and high capacity.     

Ion-induced conformational and stability changes in Nereis sarcoplasmic calcium binding protein: evidence that the APO state is a molten globule

Nereis sarcoplasmic Ca(2+)-binding protein (NSCP) is a calcium buffer protein that binds Ca(2+) ions with high affinity but is also able to bind Mg(2+) ions with high positive cooperativity. We investigated the conformational and stability changes induced by the two metal ions. The thermal reversible unfolding, monitored by circular dichroism spectroscopy, shows that the thermal stability is maximum at neutral pH and increases in the order apo < Mg(2+) < Ca(2+). The stability against chemical denaturation (urea, guanidinium chloride) studied by circular dichroism or intrinsic fluorescence was found to have a similar ion dependence. To explore in more detail the structural basis of stability, we used the fluorescent probes to evaluate the hydrophobic surface exposure in the different ligation states. The apo-NSCP exhibits accessible hydrophobic surfaces, able to bind fluorescent probes, in clear contrast with denatured or Ca(2+)/Mg(2+)-bound states. Gel filtration experiments showed that, although the metal-bound NSCP has a hydrodynamic volume in agreement with the molecular mass, the volume of the apo form is considerably larger. The present results demonstrate that the apo state has many properties in common with the molten globule. The possible factors of the metal-dependent structural changes and stability are discussed.     

Interaction analysis of chimeric metal-binding green fluorescent protein and artificial solid-supported lipid membrane by quartz crystal microbalance and atomic force microscopy

Non-specific adsorption and specific interaction between a chimeric green fluorescent protein (GFP) carrying metal-binding region and the immobilized zinc ions on artificial solid-supported lipid membranes was investigated using the quartz crystal microbalance technique and the atomic force microscopy (AFM). Supported lipid bilayer, composed of octanethiol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid)succinyl] (NTA-DOGS)-Zn2+, was formed on the gold electrode of quartz resonator (5 MHz). Binding of the chimeric GFP to zinc ions resulted in a rapid decrease of resonance frequency. Reversibility of the process was demonstrated via the removal of metal ions by EDTA. Nanoscale structural orientation of the chimeric GFP on the membrane was imaged by AFM. Association constant of the specific binding to metal ions was 2- to 3-fold higher than that of the non-specific adsorption, which was caused by the fluidization effect of the metal-chelating lipid molecules as well as the steric hindrance effect. This infers a possibility for a further development of biofunctionalized membrane. However, maximization is needed in order to attain closer advancement to a membrane-based sensor device.     

Thermal stability of calmodulin and mutants studied by (1)H-(15)N HSQC NMR measurements of selectively labeled [(15)N]Ile proteins

Calmodulin, the Ca(2+)-dependent activator of many cellular processes, contains two well-defined structural domains, each of which binds two Ca(2+) ions. In its Ca(2+)-free (apo) form, it provides an attractive model for studying mechanisms of protein unfolding, exhibiting two separable, reversible processes, indicating two structurally autonomous folding units. (1)H-(15)N HSQC NMR in principle offers a detailed picture of the behavior of individual residues during protein unfolding transitions, but is limited by the lack of dispersion of resonances in the unfolded state. In this work, we have used selective [(15)N]Ile labeling of four distinctive positions in each calmodulin domain to monitor the relative thermal stability of the folding units in wild-type apocalmodulin and in mutants in which either the N- or C-domain is destabilized. These mutations lead to a characteristic perturbation of the stability (T(m)) of the nonmutated domain relative to that of wild-type apocalmodulin. The ability to monitor specific (15)N-labeled residues, well-distributed throughout the domain, provides strong evidence for the autonomy of a given folding unit, as well as providing accurate measurements of the unfolding parameters T(m) and DeltaH(m). The thermodynamic parameters are interpreted in terms of interactions between one folded and one unfolded domain of apocalmodulin, where stabilization on the order of a few kilocalories per mole is sufficient to cause significant changes in the observed unfolding behavior of a given folding unit. The selective (15)N labeling approach is thus a general method that can provide detailed information about structural intermediates populated in complex protein unfolding processes.     

Volume and free energy of folding for troponin C C-domain: linkage to ion binding and N-domain interaction

Troponin C (TnC) is an 18-kDa acidic protein of the EF-hand family that serves as the trigger for muscle contraction. In this study, we investigated the thermodynamic stability of the C-domain of TnC in all its occupancy states (apo, Mg (2+)-, and Ca (2+)-bound states) using a fluorescent mutant with Phe 105 replaced by Trp (F105W/C-domain, residues 88-162) and (1)H NMR spectroscopy. High hydrostatic pressure was employed as a perturbing agent, in combination with urea or without it. On the basis of changes in Trp emission, the C-domain apo state was denatured by pressure (in the range of 1-1000 bar) in the absence of urea. The fluorescence data were corroborated by following the changes in the (1)H NMR signal of Histidine 128. Addition of Ca (2+) or Mg (2+) increased the C-domain stability so that complete denaturation was attained only by the combined use of high hydrostatic pressure and either 7-8 M or 1.5-2 M urea, respectively. The (1)H NMR spectra in the presence of Ca (2+) was typical of a highly structured protein and allowed us to follow the changes in the local environment of several amino-acid residues as a function of pressure at 4 M Urea. Different residues presented different volume changes, but those that are in the hydrophobic core portrayed values very similar to that obtained for tryptophan 105 as measured by fluorescence, indicating that it is indeed a good probe for the overall tertiary structure. From these experiments, we calculated the thermodynamic parameters (Delta G degrees atm and Delta V) that govern the folding of the C-domain in all its possible physiological states and constructed a thermodynamic cycle. Furthermore, a comparison of the volume and free-energy changes of folding of isolated C-domain with those of intact TnC (F105W) revealed that the N-domain has little effect on the structure of the C-domain, even in the presence of Ca (2+). The volume and free-energy diagrams reveal a landscape of different conformations from the less structured, denatured apo form to the highly structured, Ca (2+)-bound form. The large change in folding free energy of the C-domain that takes place when Ca (2+) binds may explain the much higher Ca (2+) affinity of sites III and IV, 2 orders of magnitude higher than the affinity of sites I and II.     

Structural and energetic determinants of apo calmodulin binding to the IQ motif of the Na(V)1.2 voltage-dependent sodium channel

The neuronal voltage-dependent sodium channel (Na(v)1.2), essential for generation and propagation of action potentials, is regulated by calmodulin (CaM) binding to the IQ motif in its α subunit. A peptide (Na(v)1.2(IQp), KRKQEEVSAIVIQRAYRRYLLKQKVKK) representing the IQ motif had higher affinity for apo CaM than (Ca(2+))(4)-CaM. Association was mediated solely by the C-domain of CaM. A solution structure (2KXW.pdb) of apo (13)C,(15)N-CaM C-domain bound to Na(v)1.2(IQp) was determined with NMR. The region of Na(v)1.2(IQp) bound to CaM was helical; R1902, an Na(v)1.2 residue implicated in familial autism, did not contact CaM. The apo C-domain of CaM in this complex shares features of the same domain bound to myosin V IQ motifs (2IX7) and bound to an SK channel peptide (1G4Y) that does not contain an IQ motif. Thermodynamic and structural studies of CaM-Na(v)1.2(IQp) interactions show that apo and (Ca(2+))(4)-CaM adopt distinct conformations that both permit tight association with Na(v)1.2(IQp) during gating.     

Influence of metal ions on structure and catalytic activity of papain

Papain is an endoprotease belonging to cysteine protease family. The catalytic activity of papain in presence of two different metal ions namely zinc and cadmium has been investigated. Both the metal ions are potent inhibitors of the enzyme activity in a concentration dependent manner. The enzyme loses 50% of its activity at 2 x 10(-4) M of CdCl2 and 4 x 10(-4) M of ZnCl2. It is completely inactivated above 1 x 10(-3) M concentration of either ZnCl2 or CdCl2. Of the two metal ions zinc with a ki value of 5 x 10(-5) M is a more potent inhibitor than cadmium which has a ki value of 8 x 10(-5) M. Both the metal ions have higher affinity for active site than the substrate. At concentrations above 1 x 10(-2) M of metal ions the inhibition is not reversible. Calorimetric studies showed decreased thermal stability of papain upon binding of these metal ions. Far UV circular dichroic spectral data showed only small changes in the beta-structure content upon binding of these metal ions. These data are also supported by decrease in the apparent thermal transition temperature of papain by 5 degrees C upon binding of metal ions indicating destabilization of the papain molecule. The mechanism of both partial and complete inactivation of papain in presence of these two metal ions both at lower and higher concentration has been explained.     

Perforin lytic activity is controlled by calreticulin

The components within cytotoxic lymphocyte granules are responsible for a significant fraction of T and NK cell-mediated death. Perforin is stored in these granules together with calreticulin. Calreticulin has long been recognized as a chaperone protein of the endoplasmic reticulum (ER) and is the only resident ER protein to be found in the cytotoxic granules. Here we implicate a role for calreticulin in killing and report that it controls osmotic lysis mediated by purified perforin. Calreticulin, at a concentration of 2.2 x 10-7 M, completely blocked perforin-mediated lysis. Inhibition was stable and held over 5 h. Recombinant calreticulin, at a concentration of 8. 8 x 10-7 M, also blocked lysis, indicating the inhibition was due to calreticulin and not a copurifying protein in the native calreticulin preparations. Using calreticulin domain fragments (expressed as GST fusion proteins), we found inhibitory activity in the high-capacity calcium-binding C-domain, which does not bind perforin. The N- or P-domains, which can bind perforin, were unable to block lysis. The inhibition of lysis was independent of granzyme inactivation or the ability of calreticulin to sequester calcium. Our data indicate that calreticulin regulation of perforin-mediated lysis probably occurs without direct interaction with perforin. We propose a novel model in which calreticulin stabilizes membranes to prevent polyperforin pore formation.     

Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones

We have used multimode fluorescent speckle microscopy (FSM) and correlative differential interference contrast imaging to investigate the actin-microtubule (MT) interactions and polymer dynamics known to play a fundamental role in growth cone guidance. We report that MTs explore the peripheral domain (P-domain), exhibiting classical properties of dynamic instability. MT extension occurs preferentially along filopodia, which function as MT polymerization guides. Filopodial bundles undergo retrograde flow and also transport MTs. Thus, distal MT position is determined by the rate of plus-end MT assembly minus the rate of retrograde F-actin flow. Short MT displacements independent of flow are sometimes observed. MTs loop, buckle, and break as they are transported into the T-zone by retrograde flow. MT breakage results in exposure of new plus ends which can regrow, and minus ends which rapidly undergo catastrophes, resulting in efficient MT turnover. We also report a previously undetected presence of F-actin arc structures, which exhibit persistent retrograde movement across the T-zone into the central domain (C-domain) at approximately 1/4 the rate of P-domain flow. Actin arcs interact with MTs and transport them into the C-domain. Interestingly, although the MTs associated with arcs are less dynamic than P-domain MTs, they elongate efficiently as a result of markedly lower catastrophe frequencies.     

Role of calnexin, calreticulin, and endoplasmic reticulum mannosidase I in apolipoprotein(a) intracellular targeting

Apolipoprotein(a) [apo(a)] is a component of atherogenic lipoprotein(a) [Lp(a)]. Differences in the extent of endoplasmic reticulum (ER) associated degradation (ERAD) of apo(a) allelic variants contribute to the >1000-fold variation in plasma Lp(a) levels. Using human apo(a) transgenic mouse hepatocytes, we analyzed the role of the ER chaperones calnexin (CNX) and calreticulin (CRT), and ER mannosidase I in apo(a) intracellular targeting. Co-immunoprecipitation and pulse-chase analyses revealed similar kinetics of apo(a) interaction with CNX and CRT, peaking 15-30 min after apo(a) synthesis. Trapping of apo(a) N-linked glycans in their monoglucosylated form, by posttranslational inhibition of ER glucosidase activity with castanospermine (CST), enhanced apo(a)-CNX/CRT interaction and prevented both apo(a) secretion and ERAD. Delay of CST addition until 20 or 30 min after apo(a) synthesis [when no apo(a) had yet undergone degradation or Golgi-specific carbohydrate modification] allowed a portion of apo(a) to be secreted or degraded. These results are consistent with a transient apo(a)-CNX/CRT association and suggest that events downstream of CNX/CRT interaction determine apo(a) intracellular targeting. Inhibition of ER mannosidase I with deoxymannojirimycin or kifunensine had no effect on apo(a) secretion, but inhibited proteasome-mediated apo(a) ERAD even under conditions where apo(a)-CNX/CRT interaction was prevented. These results suggest a role for an additional, mannose-specific, ER lectin in targeting secretory proteins to the proteasome for destruction.     

Role of metals in the biological activity of Clostridium botulinum neurotoxins

Clostridium botulinum neurotoxins are the most potent toxins to humans and cause paralysis by blocking neurotransmitter release at the presynaptic nerve terminals. The toxicity involves four steps, viz., binding to neuronal cells, internalization, translocation, and catalytic activity. While the catalytic activity is a zinc endopeptidase activity on the SNARE complex proteins, the translocation is believed to be a pH-dependent process allowing the translocation domain to change its conformation to penetrate the endosomal membrane. Here, we report the crystal structures of botulinum neurotoxin type B at various pHs and of an apo form of the neurotoxin, and discuss the role of metal ions and the effect of pH variation in the biological activity. Except for the perturbation of a few side chains, the conformation of the catalytic domain is unchanged in the zinc-depleted apotoxin, suggesting that zinc's role is catalytic. We have also identified two calcium ions in the molecule and present biochemical evidence to show that they play a role in the translocation of the light chain through the membrane.     

Single-Molecule Folding Mechanisms of the apo- and Mg2+-Bound States of Human Neuronal Calcium Sensor-1

Neuronal calcium sensor-1 (NCS-1) is the primordial member of a family of proteins responsible primarily for sensing changes in neuronal Ca²⁺ concentration. NCS-1 is a multispecific protein interacting with a number of binding partners in both calcium-dependent and independent manners, and acting in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca²⁺-activated state of NCS proteins, little is known about the conformational dynamics of the Mg²⁺-bound and apo states, both of which are populated, at least transiently, at resting Ca²⁺ conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg²⁺ and in the absence of divalent ions. Under tension, the Mg²⁺-bound state of NCS-1 unfolds and refolds in a three-state process by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions were reconstructed through hidden Markov model analysis. Unlike what has been observed with the Ca²⁺-bound state, the presence of Mg²⁺ allows both the N- and C-domain to fold through all-or-none transitions with similar refolding rates. In the absence of divalent ions, NCS-1 unfolds and refolds reversibly in a two-state reaction involving only the C-domain, whereas the N-domain has no detectable transitions. Overall, the results allowed us to trace the progression of NCS-1 folding along its energy landscapes and provided a solid platform for understanding the conformational dynamics of similar EF-hand proteins.     

Mechanism of mutant calreticulin-mediated activation of the thrombopoietin receptor in cancers

Myeloproliferative neoplasms (MPNs) are frequently driven by mutations within the C-terminal domain (C-domain) of calreticulin (CRT). CRT_Del52 and CRT_Ins5 are recurrent mutations. Oncogenic transformation requires both mutated CRT and the thrombopoietin receptor (Mpl), but the molecular mechanism of CRT-mediated constitutive activation of Mpl is unknown. We show that the acquired C-domain of CRT_Del52 mediates both Mpl binding and disulfide-linked CRT_Del52 dimerization. Cysteine mutations within the novel C-domain (C400A and C404A) and the conserved N-terminal domain (N-domain; C163A) of CRT_Del52 are required to reduce disulfide-mediated dimers and multimers of CRT_Del52. Based on these data and published structures of CRT oligomers, we identify an N-domain dimerization interface relevant to both WT CRT and CRT_Del52. Elimination of disulfide bonds and ionic interactions at both N-domain and C-domain dimerization interfaces is required to abrogate the ability of CRT_Del52 to mediate cell proliferation via Mpl. Thus, MPNs exploit a natural dimerization interface of CRT combined with C-domain gain of function to achieve cell transformation.